Semaphorin4D Induces Inhibitory Synapse Formation by Rapid Stabilization of Presynaptic Boutons via MET Coactivation

Abstract

Changes in inhibitory connections are essential for experience-dependent circuit adaptations. Defects in inhibitory synapses are linked to neurodevelopmental disorders, but the molecular processes underlying inhibitory synapse formation are not well understood. Here we use high-resolution two-photon microscopy in organotypic hippocampal slices from GAD65-GFP mice of both sexes to examine the signaling pathways induced by the postsynaptic signaling molecule Semaphorin4D (Sema4D) during inhibitory synapse formation. By monitoring changes in individual GFP-labeled presynaptic boutons, we found that the primary action of Sema4D is to induce stabilization of presynaptic boutons within tens of minutes. Stabilized boutons rapidly recruited synaptic vesicles, followed by accumulation of postsynaptic gephyrin and were functional after 24 h, as determined by electrophysiology and immunohistochemistry. Inhibitory boutons are only sensitive to Sema4D at a specific stage during synapse formation and sensitivity to Sema4D is regulated by network activity. We further examined the intracellular signaling cascade triggered by Sema4D and found that bouton stabilization occurs through rapid remodeling of the actin cytoskeleton. This could be mimicked by the actin-depolymerizing drug latrunculin B or by reducing ROCK activity. We discovered that the intracellular signaling cascade requires activation of the receptor tyrosine kinase MET, which is a well known autism risk factor. By using a viral approach to reduce MET levels specifically in inhibitory neurons, we found that their axons are no longer sensitive to Sema4D signaling. Together, our data yield important insights into the molecular pathway underlying activitydependent Sema4D-induced synapse formation and reveal a novel role for presynaptic MET at inhibitory synapses.

Experience-dependent inhibitory plasticity is mediated by CCK+ basket cells in the developing dentate gyrus

“…Another hypothesis is that axon terminals from INs are added but the maturation of the postsynaptic counterpart is delayed. The later hypothesis is consistent with in vitro work showing that VGAT clustering precedes that of Gephyrin (Dobie & Craig, 2011;Frias et al, 2019). Future work will be needed to distinguish between these two non-exclusive hypotheses.…”

Reduction of Dendritic Inhibition in CA1 Pyramidal Neurons in Amyloidosis Models of Early Alzheimer’s Disease

“…Organotypic hippocampal slice cultures were prepared from postnatal day 6-8 old GAD65-GFP mice as previously described [24,25]. In short, mice of both sexes were decapitated and their brains were placed in ice cold Gey's Balanced Salt Solution (GBSS, in mM: 137 NaCl, 5 KCl, 1.5 CaCl2, 1 MgCl2, 0.3 MgSO4, 0.2 KH2PO4, 0.85 Na2HPO4).…”

“…24 h A␤ oligomer treatment reduces dendritic inhibition in organotypic hippocampal slices. A) Example traces of action potentials after current injections.…”

Amyloid-β Oligomers Induce Only Mild Changes to Inhibitory Bouton Dynamics

“…1B ), as previously described [ 14, 30–32 ]. We distinguished two populations of boutons: persistent boutons which were present during the entire recording period, and non-persistent boutons which are present only in a subset of time points (see methods) [ 31 ]. 24 h Aβ treatment did not affect overall inhibitory bouton density ( Fig.…”

“…Bouton dynamics are determined by monitoring the same axon over all time points and determining bouton occurrence over time. We categorized boutons as described before ([ 31 ], see also Supplementary Figure 1 ). Boutons are divided in persistent boutons, which are present during the entire imaging period and non-persistent boutons, which are present only in a subset of time points.…”